This invention is directed to methods for amplifying desired nucleotide sequences and assaying the nucleotides produced by such amplifications.
Recent advances in the general field of molecular biology have made it possible to detect specific genes of clinical and commercial importance. The use of nucleic acid hybridization assays as a research tool for the detection and identification of a unique deoxyribonucleic acid (DNA) sequence or a specific gene in a complete DNA, a mixture of DNA's, or a mixture of DNA fragments have made it possible to diagnose human disease at the genetic level.
The most common techniques for detecting a specific gene sequence are hybridization-based assays. A specific nucleotide sequence of probe is marked with a detectable label, typically a radioactive label (isotopic) or chemical modification (non-isotopic). The detectable label is combined with the nucleic acid sample of interest, either in situ as part of intact cells or as isolated DNA or RNA fragments. The hybridization conditions should be those which allow the probe to form a specific hybrid with its complementary DNA or RNA target while not becoming bound to non-complementary DNA or RNA molecules. The target sample sequence can be either free in solution or immobilized on a solid substrate. The probe's detectable label provides a means for determining whether hybridization has occurred and, thus, for detecting the DNA or RNA target.
The recent advances in automated nucleic acid oligonucleotide (ribo- and deoxyribo-) syntheses and the polymerase chain reaction (ICR) method of DNA amplification have increased the power and sensitivity of nucleic acid hybridization assays. PCR is an in vitro method for the enzymatic synthesis of specific DNA sequences, using two deoxyoligonucleotide primers that hybridize to opposite strands and flank the specific target region of DNA that is to be amplified. The use of automated thermal cyclers allows a repetitive series of reaction steps involving template denaturation, primer annealing and the extension of the annealed primers by DNA polymerase resulting in the exponential accumulation of the specific target region of DNA whose termini are defined by the 5' end of the primers.
PCR technique is an extremely powerful method for amplifying nucleic acid sequences, however the detection of the amplified material may require additional manipulation and subsequent handling of the PCR products to determine whether the target region of DNA is present. For example, removal of labeled probe that has not come into contact with the target sequence significantly complicates typical hybridization assays. A more useful probe technique would minimize the number of additional handling steps currently required for the detection of the amplified material. Ideally, such a technique would combine the amplification and detection steps into a homogeneous system, thereby eliminating the need for a post amplification phase separation of target-contracted and target-non-contacted probe prior to signal detection.
Prior art nucleotide detection strategies generally fall into two categories. The first is amplification of the target material to improve the efficacy of conventional detection methods. Livak et al., PCR Methods and Applications, 4:357 (1995), offers an example of this strategy by teaching the use of a reporter fluorescent dye and a quencher fluorescent dye attached to the 5' and 3' ends of an oligonucleotide probe. As the polymerase moves along the target DNA sequence in a 3' direction, its 5' nuclease activity first displaces and then cleaves the oligonucleotide probe, separating the reporter from the quencher. Thus, presence of target DNA sequence may be measured by detecting fluorescence of the reporter dye. Since this method depends on the 5' nuclease activity of the polymerase, significant constraints are placed on the design of probes that can be used. For example, the label must be attached to DNA and the probe must be designed to allow cleavage from the 5' end. Moreover, since one enzyme is being required to provide both polymerase and nuclease activity, it is not possible to independently select or optimize those events.
The second category features methods that rely on amplification of the probe signal produced by the target sequence, instead of amplifying the target directly. These methods require significant handling steps and are directed to an end point analysis as opposed to a kinetic, real time determination of target sequence presence. For example, U.S. Pat. Nos. 4,876,187 and 5,011,769, Duck et al., Bio Techniques, 9:142 (1990) and Bekkaoui et al., Bio Techniques, 2:240 (1996) disclose a cycling probe method that employs probes comprising RNA, preferably DNA:RNA:DNA chimeras. The reaction is carried out isothermally, using a temperature at which the chimeric probes will anneal to the target DNA. An enzyme such as RNase H is used to digest the RNA portion of the probe and generate shorter, labeled oligonucleotides that dissociate at the reaction temperature. The target DNA sequence is then available for hybridization with another probe and, after a number of cycles, sufficient label has been generated to collect and detect. In general, these methods rely on immobilizing a portion of the label to allow for phase separation and signal recovery and measurement. Since these systems require amplification of the probe signal, they are designed to be used as an alternative to conventional target amplification strategies and require isothermal conditions. Further, the methods rely on phase separation for detection of the label, and thus, are not directed to homogenous systems. Also, the choice of probe design is limited because the nuclease activity of polymerases could attack the DNA portion of a chimeric probe, generating false signal.
The difficulties posed in providing accurate detection of alleles or mutants that differ in sequence from related strains by as little as a single base exemplify the deficiencies of the prior art. Methods that use selective digestions with restriction enzymes followed by electrophoretic separation require substantial post-amplification handling. Homogeneous fluorogenic probes such as those described by Livak, supra, have only limited capacity to detect rare sequences despite being optimized for such discrimination. Similarly, probe amplification strategies such as taught in U.S. Pat. No. 5,660,988 to Duck et al. also are limited. These latter prior art methods suffer from drawbacks in at least two areas.
First, both target amplification and probe amplification strategies exploit the difference in annealing force of the probe between perfectly matched and non-perfectly matched sequences to generate different signal intensities with the respective sequences. The annealing force acting between a probe and its target sequence is the sum of the interactive forces acting between the individual base pairs. Signal intensity is thus related to the aggregate of all the interactions of the base pairs. Mismatch at a single base affects the interactive force in a graded manner. Strategies solely relying upon the difference in annealing force between target nucleic acid sequences are of limited usefulness for the detection and measurement of a nucleic acid sequence present in low concentration relative to the corresponding sequence present in high concentration.
Second, target amplification strategies entail the risk that rare sequences may be lost or drop-out during amplification where another sequence, present in significant excess over the rare sequence, is also being amplified. Consumption of substrate and production of inhibitory byproducts, base misincorporation in the dominant sequence, and generation of background signal from the dominant sequence all contribute to limiting detection of rare sequences.
Qualitative and quantitative detection of variable nucleic acid targets is also a limitation of prior art methods. Because signal generation by prior art detection strategies are affected by the annealing force between the probe and nucleic acid target, sequence mismatches will result in diminished signal generation. Quantitative assays may result in underestimation of the nucleic acid target while qualitative assays may provide false negative results. (Science Jun. 6, 1997, 276:1488-91).
To avoid these difficulties, probeless detection strategies have been devised. Higuchi and others have devised techniques based on the change in fluorescent signal generated by fluorogenic dyes upon binding to double-stranded DNA produced during the amplification reaction. (Biotechnology 10(4):412-417, April 1992) Nazarenko and others have incorporated a hair-pin structure labeled with energy transfer fluorophores at the 5' end of a primer used in PCR-based amplification. The primers are designed so that incorporation of the labeled primer into the amplification product relieves fluorescence quenching resulting in a detectable signal.
Strategies directed toward the inhibition of amplification of the target nucleic acid sequence have been devised. PNA (peptide nucleic acid) probes have been designed to compete with PCR primers under conditions where the annealing force of the fully complementary PNA probe is sufficient to prevent primer annealing and amplicon formation. Mismatched PNA probes, however, have insufficient annealing force to prevent primer annealing and amplicon formation. Kyger EM et al., "Detection of the hereditary hemochromatosis gene mutation by real-time fluorescence polymerase chain reaction and peptide nucleic acid clamping", Anal Biochem, 260:2, 142-8 (1998).
Probeless detection strategies have limited specificity and sensitivity because of the inability of these methods to distinguish amplification products generated by priming of non-complementary nucleic acid sequences or the generation of primer dimers. There remains a need for the development of strategies which satisfy the need for accurate qualitative and quantitative detection of the nucleic acid target while overcoming the difficulties caused by generation of non-specific amplification products.
Accordingly, there remains a need for improved amplification strategies. There is also a need for amplificaton strategies capable of providing homogenous detection of nucleic acid sequences, wherein the detection method is integrated into and compatible with amplification of the nucleotide. This is a further need for detection of amplification products that allow the use of more versatile probe designs. There is also a need for nucleotide amplification and detection methods that permit accurate discrimination of mutant and rare sequences. This invention satisfies these and other needs.